The Role Of H₂S In Melatonin-Induced Salt Tolerance In Cucumber

Salt stress is one of the major abiotic stresses that cause crop yield reduction and severely restrict the sustainable development of vegetable crops.Cucumber,as an important vegetable crop in China,is very sensitive to salinity during the seedling stage and is highly susceptible to serious losses in its production.Melatonin（MT）,a novel plant growth regulator,can enhance the tolerance of cucumber to salt stress.However,its in-depth molecular mechanism has not been fully elucidated.Hydrogen sulfide（H₂S）,as the third gas signaling molecule after nitric oxide（NO）and carbon monoxide（CO）,plays an important role in regulating plant response to salt stress.However,the relationship between melatonin and H₂S has not been revealed yet.In this study,we investigated the role of H₂S in the regulation of melatonin in response to salt stress in different tissues and its interrelationship with NO and MAPK cascade pathways using cucumber ’Xinchun Ⅳ’ as experimental material.The main results were as follows.1.Both MT or NaCl treatment induced H₂S production in leaves and roots,and leaf H₂S content peaked at 4 h and gradually decreased to CK level from 6 to 24 h,while root H₂S content peaked at 3 h and slightly decreased but maintained at a certain level from 4 to 24 h.MT or NaCl treatment caused an increase in the activity and gene expression of LCD（L-cysteine desulfhydrase）and DCD（D-cysteine desulfhydrase）enzymes,the key enzymes for H₂S synthesis,in both leaves and roots,but they were inhibited by H₂S scavenger（HT）,H₂S synthesis inhibitor（PAG）and melatonin biosynthesis inhibitor（p-CPA）.It is suggested that both MT and NaCl treatments rapidly activate the L/DCD pathways,inducing the production of large amounts of H₂S,while endogenous melatonin may act upstream of H₂S.However,NaCl also induces the expression of other genes on the root H₂S synthesis pathway,suggesting that the L/DCD pathway is not the main pathway for H₂S induction by NaCl treatment in roots.2.MT + NaCl treatment significantly increased photosynthetic gas exchange parameters（Et,Pn,Gs）and chlorophyll fluorescence parameters（Fv/Fo,Fv/Fm,ETR,Yiled and qP）and decreased Ci and qN in cucumber leaves compared to NaCl treatment.MT + NaCl + HT,MT + NaCl + PAG,MT + NaCl + c PTIO（NO scavenger）treatments significantly suppressed the changes in photosynthetic efficiency induced by MT +NaCl treatment.It indicates that the regulation of photosynthesis in cucumber under salt stress by melatonin needs to be dependent with H₂S and NO.3.NaCl treatment leads to a significant accumulation of ROS(O₂^·-,·OH and H₂O₂)in cucumber leaves and roots compared to CK,but it is weakened by MT treatment.HT,PAG,c PTIO,and U0126（MAPKK inhibitor）instead exacerbated oxidative damage in leaves and roots compared with MT + NaCl treatment.In addition,MT + NaCl treatment significantly up-regulated the expression levels of Cu-Zn SOD,Mn SOD,POD2,CAT1,APX,DHAR,and GR and increased the activities of SOD,POD,CAT,APX,DHAR,and GR in leaves and roots compared with NaCl treatment,indicating that MT has a protective effect against antioxidant defense in cucumber leaves and roots.However,this protective effect is disrupted by HT,PAG,c PTIO and U0126.It can be seen that H₂S,NO and MAPK are involved in the regulation of melatonin on the antioxidant defense of cucumber under salt stress.4.MT + NaCl treatment increased NaCl-induced increase in leaf and root LCD,DCD enzyme activity and gene expression levels,and induced high production of H₂S,but was significantly inhibited by c PTIO,which was not affected by MAPKK inhibitor U0126.On the other hand,MT + NaCl treatment induced more NO production,but would be significantly inhibited by HT,while MT + NaCl + U0126 treatment showed no significant difference in NO content compared to MT + NaCl treatment.It indicates that H₂S produced during melatonin regulation of salt stress in cucumber has a reciprocal effect with NO,and the MAPK cascades may be located downstream of H₂S.5.Compared with CK,MT + NaCl treatment induced elevated expression of MAPK3,MAPK4,MAPK6,and MAPK9 in leaves and roots,but was significantly inhibited by HT and c PTIO,and MAPK6 was most significantly inhibited.Immunoprecipitation analysis showed that MT（100 μM）and NaCl（150 μM）treatments significantly activated MAPK6 kinase at 46 k Da and would be inhibited by 100 μM U0126.MT（100 μM）and NaCl（150 μM）significantly induced high MAPK6 protein expression in roots at 1 h,suggesting that MAPK6 protein expression was significantly affected by MT and NaCl with dose-and time-effects.In contrast,MT + NaCl treatment induced MAPK6 protein expression at 0.5 h and persisted until 4 h,but would be almost completely inhibited by HT and c PTIO at 2 and 3 h.The above results suggest that the MAPK cascade pathway is located downstream of H₂S and NO and plays a role in the regulation of salt stress in cucumber by melatonin.